Pharmaceutical compositions

ABSTRACT

An inhalent allergen composition comprises an inhalent allergen and a saponin adjuvant. The inhalent allergen may be an extract of a grass pollen, such as rye grass, or a weed, such as ragweed. The saponin adjuvant may be an extract from the bark of the tree Quillaja Molina, and known as Quil-A.

This invention relates to pharmaceutical compositions, their use in thetherapy of allergic humans, and to a process for their preparation.

Saponin adjuvants are a known class of adjuvants which have been usedcommercially to adjuvant Foot and Mouth Disease vaccines for animals.

It has now surprisingly been found that such adjuvants are useful ininhalent allergen compositions, which compositions may be used in thedesensitisation therapy of humans allergic to inhalent allergens.

Accordingly the present invention provides a pharmaceutical composition,which composition comprises an inhalent allergen and a saponin adjuvantoptionally in combination with a pharmaceutically acceptable carrier.

The adjuvant is suitably in the form of a physical admixture with theallergen in the composition of the invention, although under certainconditions some degree of conjugation or covalent bonding between theadjuvant and the allergen can occur.

Such conjugation suitably occurs by first preparing a modified form ofthe adjuvant in which the functional groups of the adjuvant areactivated, and linking this activated form to the allergen. Suitableactivating agents for the adjuvant are carboxyl activating agents wellknown to those skilled in the art. A particularly useful activatingagent is N-(3-dimethylaminopropyl)-N¹ -ethyl carbodiimide (CDI), whichmay be used in combination with N-hydroxysuccinimide.

Both physical admixture and conjugation of the active ingredients arewithin the scope of the compositions of the invention.

The inhalent allergen for use in this invention is suitably in the formof an extract of whole allergen. Suitable whole allergens from which theextract can be obtained may be chosen from any allergen which exerts itseffect via inhalation. By way of illustration pollens, such as grasspollens, for example rye; weeds, such as ragweed; house dust mites; anddander; are all suitable. These extracts of allergen are water soluble.More suitably the allergen is an extract of a grass pollen.

The extraction of the allergenic material may be carried out by treatingthe whole allergen with a suitable solvent, usually aqueous. Theallergenic extract may then be purified by removing contaminants e.g. bydialysis, precipitation or gel filtration. In another useful extractionprocedure, the whole allergen or an aqueous extract thereof, is treatedwith aqueous phenol and the allergenic extract is recovered from thephenol phase.

It will of course be appreciated that it is well known in the art thatsuch allergens may be modified prior to use to achieve an optimisationof properties. By way of example, a glutaraldehyde modified allergen maybe used, such materials being described fully in U.K. Pat. No.1,282,163.

Also of course other known formulation techniques may be used with theallergens for use in this invention--they may for example be adsorbed(modified or unmodified) onto tyrosine, as fully described in U.K. Pat.Nos. 1,377,074 and 1,492,973.

As noted above, saponin adjuvants are a known class of adjuvants readilyrecognisable to the skilled man. These adjuvants have been usedextensively for at least twenty years without any suggestion that theymight be useful in the particular manner referred to herein, namely topotentiate the antibody response to inhalent allergens.

The saponin adjuvants are natural products widely distributed in, forexample, plant families. They are generally characterised by a number ofcommon properties, such as the ability to foam in aqueous solution,hemolytic activity, toxicity for fish, and complexing with cholesterol.Their chemical structures are, in many cases, not fully elucidated, butsaponins can be characterised by having a steroid or triterpenoid moietyattached to a carbohydrate moiety (see for example Acta Vet Scan Supp69, 1-40 (1978)).

It will be appreciated that, as is normally the case when administrationto humans is being considered, the purity of the materials administeredis important. One commercially available saponin adjuvant of good purityis that well known by the name Quil-A, which is available from Superfosa/s, Frydenlundsvej 30, DK-2950 Vedbaek, Denmark. The physical andchemical characteristics of Quil-A are conveniently set out in the tradeliterature available from Superfos, entitled Purified Saponin AdjuvantQuil-A. It is noted in particular that Quil-A may be isolated from thebark of the South American tree Quillaja Saponaria Molina, and ischaracterised chemically as a carbohydrate moiety in glycosidic linkageto the triterpenoid quillaic acid.

From the aforesaid it will be appreciated that a preferred compositionof this invention comprises an inhalent allergen and Quil-A.

In such preferred compositions, often the inhalent allergen will be agrass pollen such as rye.

The quantities of allergen used in the pharmaceutical compositions ofthe invention will be as conventional for that allergen indesensitisation therapy. By way of example the composition mightsuitably contain 0.1 to 10,000 pnu of allergen, the lower end of thisrange being more appropriate for early in the therapy, the higher end ofthis range being more appropriate for later in the therapy.

The quantities of Quil-A that can be used in the pharmaceuticalcompositions of this invention are limited really only in that too highlevels might begin to show toxic effects, and too low levels willobviously not exert a meaningful adjuvant effect.

Within these boundaries, it is believed that it is a routine matter toderive a suitable level for the particular saponin and the particularallergen, to obtain the desired adjuvant effect coupled withpharmaceutical safety. By way of example, suitable levels of saponinadjuvant might be in the range 5 to 100, more suitably 25 to 75, forexample 50 μg per injection.

The pharmaceutical compositions of this invention may be used asvaccines in the desensitisation of humans allergic to inhalentallergens. Thus the compositions will suitably be in the form ofsolutions or suspensions, in a liquid vehicle. Preferably thecompositions will be aqueous solutions or suspensions.

Usually a patient receiving treatment with such a composition isadministered a number of injections, spread over a period of weeks ordays.

The invention also provides a process for the preparation of thecompositions of this invention, which process comprises bringing intoconjunction the allergen and the saponin adjuvant.

This process is conveniently carried out merely by mixing together theallergen and the saponin adjuvant in the presence of a suitable inertsolvent, preferably water.

In a further aspect of the process of the invention, the allergen iscovalently bonded to the saponin adjuvant by, for example, preparing anactivated form of the adjuvant, as hereinbefore described, and mixingthis with the allergen.

It will of course be appreciated that the compositions of this inventionmay suitably contain as additional ingredients any of the conventionalvaccine additives, such as sodium chloride, phosphates andpreservatives.

The invention also provides a method of treating humans allergic toinhalent allergens, which method comprises administering to the suffereran effective amount of a composition according to this invention.

The following Examples illustrate the invention.

EXAMPLE I Preparation of Injectable Rye/Quil-A Compositions

A: Rye/Quil-A

Rye grass pollen extract (R.E.) (1 mg) was dissolved in phosphatebuffered saline (Bacto Haemagglutination buffer from Difco) (1 ml)containing Quil-A (50 μg).

B: Rye/Tyrosine

R.E. (1 mg) was adsorbed to L-tyrosine (40 mg) in phosphate bufferedphenol saline (1 ml).

The absorbate was prepared as follows:

Solution

(1) 24% w/v L-tyrosine in 3.8 M. HCl;

(2) 3.2 N. NaOH;

(3) Na₂ HPO₄ (11.8 g)+NaH₂ PO₄.2H₂ O (3.0 g) made up to 100 ml withwater;

(4) Phosphate buffered saline (Difco);

(5) Phenol (5 g), NaCl (8 g), Na₂ HPO₄ (0.2 g), NaH₂ PO₄.2H₂ O (1.5 g),made up to 1 liter with water and the pH adjusted to 7.0 with NaOH+HCl;

(6) R.E. was made up at twice the final required concentration in 30 mlof solution (4).

10 ml of solution (3) was added to solution (6), then solution (1) (10ml) and solution (2) (10 ml) were run into this at approximately 1ml/minute using peristaltic pumps whilst stirring vigorously. The pH wasnot allowed to vary more than 0.5 pH units.

The resulting precipitate was centrifuged for 10 minutes and washedtwice with solution (5) and resuspended with 60 ml of solution (5).

C: Rye/Tyrosine/Quil-A

To 1 ml of the B preparation was added 50 μg of Quil-A in a minimalvolume.

D: Rye

R.E. (1 mg) was dissolved in phosphate buffered saline (1 ml).

N.B. The Quil-A referred to above was obtained from Superfos a/s as asterile aqueous solution containing 1.5% Quil-A dry matter. Difco'saddress is Detroit, Mich., USA.

Immunisation Schedule

Four groups of six female mature Hartley Strain Guinea Pigs received 1ml sub-cutaneous injections of preparations A, B, C or D respectively.They were then each given a further sub-cutaneous injection of 1 ml ofphosphate buffered saline containing 100 μg of R.E. on day 28. Theanimals were bled on days 23 and 35 and sera prepared.

The sera were tested for the presence of R.E. specific haemagglutinatingantibody by the standard method of `Chemical Modification of CrudeTimothy Grass Pollen Extract. Antigenicity and Immunogenicity changesfollowing amino group modification--D M Moran and A W Wheeler, Int ArchsAllergy appl Immun 50: 693-708 (1976)`.

    ______________________________________                                                               Haemagglutination Titre                                Guinea Pig                                                                              Immunogen    -log.sub.2 from 1/2, on day                            Number    Preparation  23        35                                           ______________________________________                                        A1        A            8         13                                           A2                     7         13                                           A3                     7         11                                           A4                     7         11                                           A5                     6         10                                           A6                     5         14                                           B1        B            <1        Died                                         B2                     <1        Died                                         B3                     <1         9                                           B4                     <1        Died                                         B5                     <1         9                                           B6                     4          9                                           C1        C            7         12                                           C2                     7         11                                           C3                     8         12                                           C4                     6         13                                           C5                     7         Died                                         C6                     8         11                                           D1        D            <1         8                                           D2                     <1         6                                           D3                     <1         7                                           D4                     <1        <1                                           D5                     <1         7                                           D6                     <1        Died                                         ______________________________________                                    

Conclusion

The effectiveness of Quil-A as an adjuvant is shown in these tests.

EXAMPLE 2 Preparation of Quil-A-Rye Conjugate Composition

Samples of Quil-A (amounts shown in Table 1 below) were dissolved inN,N-dimethylformamide (DMF) (1 ml) and N-hydroxysuccinimide (HOSu) (2equiv.) added. The solution was cooled in ice-water before the additionof N-(3-dimethylaminopropyl)-N¹ -ethylcarbodiimide hydrochloride (CDI)(1 equiv.) in DMF (0.5 ml). After stirring for 15 mins at 4° C. and 30mins while warming to room temperature these solutions were added tosolutions of rye grass pollen extract (R.E.) (25 mg) in sodium boratebuffer (0.1 m, pH 8.5) (5 ml) cooled in ice-water. The mixtures werestirred at 4° C. for 15 mins then at room temperature for 3 hours and at5° C. for for 20 hours. The solutions were then dialysed against 10 mMammonium bicarbonate (3×41) and lyophilised. The residual materials werecharacterised by primary amino group (1° NH₂) analysis and aRadioallergosorbent inhibition test (RAST inhibition).

                  TABLE 1                                                         ______________________________________                                              Amounts of reagents             RAST                                    Sample                                                                              used/mg                 1° NH.sub.2 gps.                                                               redn.                                   No.   Quil-A  CDI    HOS u Yield/mg                                                                             μmole/mg                                                                           (n fold)                            ______________________________________                                        1      0      0      0     16     0.76    0                                   2      5      0.43   0.5   15     0.72    7                                   3     10      0.85   1.0   27     0.69    23                                  4     30      2.55   3.0   23     0.52    108                                 ______________________________________                                    

Haemagglutinating antibody induction in guinea pigs with Quil-A-Rye

Groups of six guinea pigs were immunised with 1 mg of sample in PBS(Difco bacto-haemagglutination buffer) given in two subcutaneous sites;and were boosted similarly on day 28 with 100 μg of sample.

    ______________________________________                                        Group   Sample No.    Immunogen                                               ______________________________________                                        A       1             Native rye extract                                      B       1             Buffer control rye extract                              C       2             Quil-A-rye                                              D       3             Quil-A-rye                                              E       4             Quil-A-rye                                              ______________________________________                                    

Antibody to rye grass pollen extract was measured by thehaemagglutination method described in Example 1 and the results areshown in Table 2. It is apparent from the results that conjugation ofthe Quil A to the rye grass pollen extract increased the propensity ofthis antigen to produce rye grass pollen specific haemagglutinatingantibody.

                  TABLE 2                                                         ______________________________________                                                    Rye specific IgG by haemagglutination                                         -log.sub.2 from 1/2, on day                                                         14        28       35                                       Group     g. pig  Titre     Titre    Titre                                    ______________________________________                                        Native    A1      5         3         6                                       rye       2       4         3         9                                       extract   3       4         4         9                                                 4       6         6         8                                                 5       0         5        10                                                 6       0         5        >12                                                Av.     3.2       4.3      >9                                       Buffer    B1      6         6        10                                       control   2       2         5         9                                       rye       3       3         4         7                                       extract   4       4         5         9                                       Sample 1  5       3         0         7                                                 6       5         4         8                                                 Av.     3.8       4          8.3                                    Sample 2  C1      8         7        11                                                 2       9         3        10                                                 3       0         7        11                                                 4       7         7         9                                                 5       0         4         7                                                 6       9         6        >12                                                Av.     5.5       5.7      >10                                      Sample 3  D1      7         5        >12                                                2       7         8        >12                                                3       0         5        11                                                 4       0         5         9                                                 5       0         7        12                                                 6       9         8        11                                                 Av.     3.8       6.3      >11.2                                    Sample 4  E1      6         6        12                                                 2       6         5         7                                                 3       9         10       >12                                                4       8         9        >12                                                5       7         8        12                                                 6       12        12       12                                                 Av.     8         8.3      >11.2                                    ______________________________________                                    

I claim:
 1. An anti-allergenic pharmaceutical composition comprising a desensitizing effective amount of an inhalent allergen and an adjuvant effective amount of a saponin adjuvant, in combination with a pharmaceutically acceptable carrier.
 2. A composition according to claim 1, in which the allergen comprises an extract of a grass pollen or a weed.
 3. A composition according to claim 1, in which the allergen is modified with glutaraldehyde and/or adsorbed onto tyrosine.
 4. A composition according to claim 1, in which the saponin adjuvant comprises Quil-A.
 5. A composition according to claim 1, in which the composition contains from 0.1 to 10,000 pnu of allergen.
 6. A composition according to claim 1, in the form of a solution or suspension in a pharmaceutically acceptable liquid vehicle.
 7. A composition according to claim 6 suitable for use as a vaccine.
 8. A composition according to claim 7, in which the concentration of saponin adjuvant is selected to provide from 5 to 100 μg of adjuvant per injection.
 9. A composition according to claim 1, comprising rye grass pollen extract and Quil-A.
 10. A method of treating humans allergic to inhalent allergens, which comprises administering to the sufferer an amount effective for desensitization to said allergen of a composition according to claim
 1. 